Method for preparing pluripotent stem cells

Abstract

An object of the present invention is to provide a method capable of inexpensively and conveniently preparing cells having pluripotency and a very low risk of tumorigenic transformation. The cells can be prepared by suspension-culturing mammalian mesenchymal stem cells such as human mesenchymal stem cells from bone marrow (hMSC-BM) and human adipose tissue-derived mesenchymal stem cells (hAT-MSC) (also referred to as “human adipose-derived stem cells [hADSC]”), 7 types of human adherent mature cells (human hepatocyte cells [hHEP cells], human umbilical vein endothelial cells [HUVEC cells], human dermal lymphatic microvascular endothelial cells [HMVEC cells], human epidermal keratinocyte cells [NHEK cells], human bronchial epithelial cells [NHBE cells], human melanocyte cells [NHEM cells], and human smooth muscle cells [UASMC cells]), and 3 types of human adherent precursor cells (human dermal fibroblast cells [NHDF cells], human skeletal muscle myoblast cells [HSMM cells], and human osteoblast cells [NHOst cells]) to form cell masses (spheroids).

What is claimed is: 
     
       1. A method for preparing a pluripotent stem cell, comprising
 spheroid-culturing mammalian mesenchymal stem cells to form a spheroid of pluripotent stem cells; and 
 confirming a pluripotency of the spheroid of the pluripotent stem cells using a detected expression of a pluripotency marker Oct3/4, Sox2, SSEA3, or TRA-1-60 as an index, 
 wherein the spheroid-culturing is performed in a physiological aqueous solution free from serum or a serum substitute. 
 
     
     
       2. The method according to  claim 1 , wherein the mammalian mesenchymal stem cells are human mesenchymal stem cells from bone marrow or human adipose tissue-derived mesenchymal stem cells. 
     
     
       3. The method according to  claim 1 , wherein the pluripotent stem cell expresses Nanog, Oct3/4, or Sox2. 
     
     
       4. The method according to  claim 2 , wherein the pluripotent stem cell expresses Nanog, Oct3/4, or Sox2.